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2007 vol. 4
Clinical/Forensics
Figure 2 Fully resolve opiates with shared product ions on (morphine/hydromorphone and codeine/hydrocodone
an Allure® PFP Propyl column.
After mass spectrometry conditions were optimized for each compound, the
resulting mass spectra were used to generate +MRM (multiple reaction mon-
itoring) methods. Since MS/MS was used, we were able to target two +MRM
transitions per compound to verify the identity of each compound. Table I
shows the +MRM transitions and the mass spectrometer conditions.
Standards contained morphine, hydromorphone, oxymorphone, codeine,
hydrocodone, oxycodone, and 6-monoacetylmorphine (6-MAM) in methanol.
The on-column concentration used for column evaluations was 250ng for all
compounds.
Although two +MRM transitions were targeted for each compound, some
compounds, such as codeine and hydrocodone, shared all monitored product
ions (Figure 1). Since these compounds have similar mass spectra, two peaks
appear in the extracted ion chromatograms. This made it necessary to separate
codeine and hydrocodone chromatographically and identify compound peaks
by retention time. Morphine and hydromorphone present the same challenge.
Of the stationary phases tested, pentafluorophenyl propyl phase (Allure® PFP
Propyl column) produced the best chromatographic separation and peak
shape. Baseline resolution of opiates that shared the same product ions was
achieved on an Allure® PFP Propyl column in a total analysis time of 7 min-
utes (Figure 2). Mobile phase gradient and composition had a significant effect
on peak shape and resolution (data not shown) and optimized analytical con-
ditions were used.
The Allure® PFP Propyl column, coupled with an LC/MS/MS, produced positive
identification of opiates while reducing sample preparation time and keeping
analysis time short. Use of the Allure® PFP Propyl column and the LC/MS/MS
method shown here can increase sample throughput and is recommended for
routine opiates analysis.
Acknowledgement
The authors wish to thank Applied Biosystems for supplying the Applied Biosystems/MDS Sciex
API 3200™ MS/MS system used for this work.
Sample: opiates
Inj.: 10µL
Conc.: 25ug/mL
Solvent: methanol
Column: Allure ® PFP Propyl
Cat.#: 9169552
Dimensions: 50mm x 2.1mm
Particle size: 5µm
Pore size: 60Å
Compound Mass
1. morphine 286*
2. oxymorphone 302
3. hydromorphone 286*
4. codeine 300**
5. 6-monoacetylmorphine 328
6. oxycodone 316
7. hydrocodone 300**
Conditions:
Mobile phase: A: 0.1% formic acid in water
B: 0.1% formic acid in 80:20,
methanol:acetonitrile
Time: %B
0.0 10
3.00 50
6.00 50
6.10 10
8.10 Stop pumps
Flow: 0.40mL/min.
Temp.: 30°C
Det.: Applied Biosystems/MDS
Sciex API 3200™ MS/MS system
Ion source: Electrospray, positive
IonSpray voltage: 5500
Gas 1: 65psi (448kPa)
Gas 2: 45psi (310kPa)
Source temp.: 600°C
LC_PH0454 LC_PH0459
Solvent
Compound CAS# Code Conc. cat.# (ea.) price
codeine 76-57-3 PTM 1,000 34000 $23
hydrocodone 34195-34-1 PTM 1,000 34002 $23
hydromorphone 71-68-1 PTM 1,000 34063 $23
morphine 6211-15-0 PTM 1,000 34006 $23
oxycodone 124-90-3 PTM 1,000 34007 $23
oxymorphone 76-41-5 PTM 1,000 34065 $23
Exempted Drug of Abuse Reference
Materials: Opiates & Metabolites
Concentration is µg/mL. Volume is 1mL/ampul.
2 3 4 5 6 7 8
4
3
2
1
7
6
5
Full ion scan
Multiple reaction
monitoring (+MRM)
transitions in Table I
Allure® PFP Propyl Columns (USP L43)
Excellent Columns for LC/MS and ELSD
Physical Characteristics:
5µm Column, 2.1mm cat. # price
50mm 9169552 $370
50mm (with Trident Inlet Fitting) 9169552-700 $385
particle size: 5µm,
spherical
pore size: 60Å
carbon load: 17%
endcap: fully endcapped
pH range: 2.5 to 7.5
temperature limit: 80°C
PTM=purge & trap grade methanol.
Guard Cartridges qty. cat. # price
10 x 2.1mm 3-pk. 916950212 $135
10 x 4.0mm 3-pk. 916950210 $135
20 x 2.1mm 2-pk. 916950222 $135
20 x 4.0mm 2-pk. 916950220 $135
For a full product listing for these columns and reference
materials, visit our website at .
* and ** indicate compounds with shared productions.